WO1996020728A1

WO1996020728A1 - Antitumor agent potentiator comprising il-6 antagonist

Info

Publication number: WO1996020728A1
Application number: PCT/JP1995/002769
Authority: WO
Inventors: Osamu Yoshida; Youichi Mizutani
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1994-12-29
Filing date: 1995-12-28
Publication date: 1996-07-11
Also published as: EP0800829A4; JP4079461B2; ES2170815T3; CN1174507A; JP2008063344A; EP0800829A1; ATE214602T1; CA2209124C; AU4356896A; EP0800829B2; DE69525971D1; AU704723B2; DE69525971T3; EP0800829B1; HK1001512A1; KR100252743B1; DE69525971T2; JP4812736B2; CN1075387C; US6086874A

Abstract

A potentiator for antitumor agents, for example, platinum compounds such as cisplatin and carboplatin, mitomycin C, etc., which comprises an interleukin-6 (IL-6) antagonist such as an antibody against IL-6, an antibody against IL-6 receptor, an antibody against protein gp130, etc.

Description

明

Field of action of antitumor agents comprising IL-16 antagonist

The present invention relates to an antitumor agent potentiator comprising interleukin 6 antagonist which assists and enhances the effect of an antitumor agent in treating a tumor.

Background art

For chemotherapy of human tumors, alkylating agents, antimetabolites, antitumor antibiotics, platinum compounds and the like have been used. If no significant therapeutic effect is observed even when these antitumor agents are used alone, a treatment method combining multiple antitumor agents has been considered (Frei, E. Ill, Cancer Res., 32). , 2593-2607, 1992). Tumor cells have various sensitivities to antineoplastic drugs, and some are known to exhibit resistance to antitumor drugs (Magrath, I., New Directions in Cancer Treatment, 1989, Springer-Verlag). Expression of multi-drug resistance (MDR) (Tsuruo, T. et al., Cancer Res., 42, 4730-4733, 1982) Decreased cellular uptake (Sherman, SE et al., Science, 230, 412, 1985), increased DNA repair activity (Borc, RF, Metabolism and Action of Anticancer Drugs. Powis, G. & Prough, R., Taylor & Francis, London, 1987, 163-193) or promotion of inactivation of antitumor agents in cells (Teicher, BA et al., Cancer Res., 46, 4379, 1986). I have. In such cases, administration of an antitumor agent alone often does not provide the expected therapeutic effect. Renal cell carcinoma is a tumor that is refractory to antitumor agents such as cisplatin, adriamycin and vinblastine (Kakehi, Y. et al., J. Urol., 139, 862- 864, 1988; Kanamaru, et al., J. Natl. Cancer Inst., 81, 844-847, 1989; Teicher, BA et al., Cancer Res., 47, 388-393, 1987). Platinum compounds with antitumor activity, such as cisplatin, bind to DNA and inhibit DNA synthesis and cell division (Pinto, AL et al., Biochica et Biophysica Acta, 780, 167-180, 1985).

肾 Glutathione-S-transferase-17Γ (GST-r) expression and the effect of cis-bratin due to increased cytoplasmic levels of substances containing sulfidyl groups on the resistance of cell carcinoma to cisplatin. It is thought that suppression, enhancement of DNA repair capacity, or activation of oncogenes such as c-myc are involved in a complex manner (Sklar, MD et al., Cancer Res., 51, 2118-2123, 1991; Mizutani, Y. et al., Cancer in press, 1994; Nakagawa, K. et al., Japan. J. Cancer Res., 79, 301-305, 1988) o

In addition, it is said that changes in the transmembrane transport ability of tumor cells result in decreased uptake of intracellular cisplatin and increase resistance to treatment with cisplatin (Richon, V. et al., Cancer Res., 47, 2056-2061, 1987; Waud, WR et al., Cancer Res., 47, 6549-6555, 1987). Daltathione, which is the most abundant substance in mammalian cells as a substance containing a sulfidyl group, has been reported to inactivate cisbratin in cells, and intracellular glutathione in some tumors. And increased levels of metallothionein (Hromas, RA et al., Cancer LETT., 34, 9-13, 1987; Taylor, DM et al., Eur. J. Cancer, 12, 249- 254, 1976).

Glutathione is tribeptidothiol, an alkylating agent It plays an important role in inactivating DNA-binding substances such as subtilin and repairing cell damage caused by them. One effect of GST-; r is to promote inactivation of the antitumor agent by binding the above antitumor agent to glutathione.

肾 Since cell carcinoma produces interleukin 6 (IL-6) and expresses IL-16 receptor (IL-16R), IL-16 plays a role in activating the growth of renal cell carcinoma (Miki, S. et al., FEBS Lett., 250, 607-610, 1989; Takenawa,, et al., J. Natl. Cancer Inst., 83, 1668-1672, 1991). ). In addition, it has been reported that serum IL-16 levels are elevated when the prognosis of patients with 肾 cell carcinoma is poor (Blay, J., et al., Cancer Res., 52, 3317-332, 1992; Tsukamoto, T., et al., J. Urol., 148, 1778-1782, 1992). However, a clear association between IL-16 and the refractory resistance of renal cell carcinoma to antitumor agents has not been established so far, and it is unknown.

IL-16 is a multifunctional site-powered protein called B-cell stimulating factor 2 or interferon 2. IL-6 was discovered as a differentiation factor involved in the activation of B lymphocyte cells (Hirano, T. et al., Nature 324, 73-76, 1986), and subsequently affected the function of various cells. It has been found to be a multifunctional site kinase (Akira, S. et al., Ad v. In Immunology 54, 1-78, 1993). I_6 transmits its biological activity on cells via two proteins.

One is IL-16R, a ligand-binding protein with a molecular weight of about 80 KD to which IL-6 binds. IL-6R exists as a soluble IL-16R (sIL-6R) mainly composed of its extracellular region, in addition to a membrane-bound type expressed through the cell membrane and expressed on the cell membrane. The other is gpl30, which has a molecular weight of approximately 130 KD and is involved in nonligand-bound signal transmission. IL-16 and IL-6R form an IL-6 ZIL-6R complex, followed by another membrane protein, gp By coupling with 130, the biological activity of IL-16 is transmitted to cells (Taga et al., J. Exp. Med., 196, 967, 1987).

Platinum compounds such as cis-bratin and antitumor agents such as mitomycin C induce apoptosis in tumor cells, whereas IL-16 suppresses apoptosis induced by antitumour agents (Kerr, J. et al., Cancer 73, 2013-2026, 1994; Sachs, L. et al., Blood 82, 15-21, 1993). In addition, antitumor agents such as cisplatin and mitomycin produce cytotoxicity in tumor-producing cells by producing free radicals (Oyanagi, Y. et al., Biochem. Pharmacol. Nakano, H. et al., Biochem. Biophys. Acta., 796, 285-293, 1984) reported that IL-6 has a free radical-degrading activity. It is known that the expression of manganese superoxide dismutase (MnSOD) is promoted, and that the 1L-6 antibody suppresses the promoted expression of Mn SOD (Ono, M., et al., Biochem. Biophys. Res. Commun., 182, 1100-1107, 1992; Dougal 1, WC et al., Endocrinology, 129, 2376-2384, 1991) ₀

However, none of these reports mentions enhancing the action of antitumor agents by blocking the biological activity of IL-16. None attempted to use IL-16 Antagonist.

Until now, antitumor agents have been used to treat tumors.However, if these drugs are used in large amounts, undesired side effects such as nausea and vomiting, 肾, hepatic dysfunction and bone marrow dysfunction may occur. On the other hand, it was sometimes dangerous to administer the required amount to sufficiently exert the antitumor effect. In addition, there are tumors resistant to treatment that are ineffective with conventional chemotherapy using antitumor agents, and the emergence of an action enhancer that increases the sensitivity of such tumors to antitumor agents has been awaited. An object of the present invention is to provide a new antitumor agent action enhancer that assists and enhances the action of an antitumor agent and increases the sensitivity of tumor cells resistant to treatment with the antitumor agent. More specifically, the present invention provides an antitumor agent action enhancer comprising IL-16 antagonist. More specifically, the present invention provides an action enhancer for a chemotherapeutic agent having an antitumor effect, comprising an IL-16 antagonist. Disclosure of the invention

The present inventors have conducted intensive studies on the effects of IL-16 antagonists on changes in the sensitivity of tumor cells to antitumor agents, and have found that IL-6 antagonists such as IL-6 antibody or IL-16R antibody. Increased the sensitivity of tumor cells to the antitumor agent, and a lower dose of the antitumor agent showed a therapeutic effect. The present inventors have found that a therapeutic effect can be exhibited by using an action enhancer containing an antagonist in combination, and have completed the present invention.o

That is, the present invention relates to an antitumor agent action enhancer comprising IL-16 antagonist. More specifically, the present invention relates to an agent for enhancing the action of a chemotherapeutic agent having an antitumor effect, which comprises an IL-16 antagonist.

As the IL-6 antagonist, an antibody against IL-6, an antibody against -6R and the like are preferable, and for example, these monoclonal antibodies are preferable. Specific examples of the monoclonal antibody include a PM-1 antibody and a humanized PM-1 antibody. Examples of the antitumor agent used in combination with the IL-16 antagonist include chemotherapeutic agents, for example, a platinum compound having antitumor activity, mitomycin C, and the like. Platinum compounds include cisplatin, carboplatin, 254-S, DWA-21 1 4R, NK-121 and the like. BRIEF DESCRIPTION OF THE FIGURES

Figures 1A and IB show kidney cells in the presence of 0, 0.1, 1 or 10 µg Zml of cisplatin and IL-6 antibody (Figure 1A) or IL-6R antibody (Figure 1B). Shows cytotoxic activity against cancer strain Caki-1. ♦ indicates only cisplatin, circle indicates coexistence of cisplatin and 0.1 と g Zml of IL-6 antibody or IL-16R antibody, indicates cisplatin and l〃g noml of IL-6 antibody or IL-6R antibody presence, ginseng represents the cytotoxic activity (%) in the presence of 1 Sea 6 antibody or IL-6R antibody of Shisuburachin and ^10/1111.

Figures 2A and 2B show the results when mitomycin C and IL-6 antibody (Figure 2A) or IL-6R antibody (Figure 2B) coexist at a concentration of 0, 0.1, 1 or 10 g Zml. Shows cytotoxic activity against renal cell carcinoma cell line Caki-1. ♦ indicates mitomycin C only, garden shows mitomycin C in the presence of 0.1 gZml IL-16 antibody or IL-6R antibody. ▲ indicates mitomycin C and 1 g / ml IL-16 In the presence of the antibody or the IL-6R antibody, the reference shows the cytotoxic activity () in the presence of the mitomycin C and 10 // g Zml of the IL-6 antibody or the IL-16R antibody.

FIGS. 3A and 3B show the results of the renal development in the presence of 0, 0.1, 1 or 10 g / ml of cisplatin and IL-6 antibody (FIG. 3A) or 1L-16R antibody (FIG. 3B). Shows cytotoxic activity against cell carcinoma cell line Caki-1 / DDP. ♦ indicates only cisplatin, 酾 indicates coexistence of cisplatin and 0.1 μg ml of IL-6 antibody or IL_6R antibody, and ▲ indicates cisplatin and 1 μg / ml IL-6 antibody or 1L-16R antibody Indicates the cytotoxic activity (%) in the co-presence of cisplatin and 10 g / ml of the IL-6 antibody or 1L-16R antibody. Figures 4A and 4B show renal cells in the presence of 0, 0.1, 1 or 10 zg Zml of cisplatin and IL-6 antibody (Figure 4A) or IL-6R antibody (Figure 4B). Fig. 4 shows cytotoxic activity against cancer cell line ACHN. ♦ indicates only cisplatin, 丽 indicates coexistence of cisplatin and 0.1 〃g Zml of IL-6 antibody or IL-16R antibody, ▲ indicates cisbratin

In the co-presence of the IL-16 or IL-6R antibody, the pareto shows the cytotoxic activity (%) in the co-presence of cisplatin and 10 gZml of the IL-6 or IL-6R antibody.

FIGS. 5A and 5B show the results of the renal development when cisplatin and IL-6 antibody (FIG. 5A) or IL-6R antibody (FIG. 5B) coexist at concentrations of 0, 0.1, 1 or 10 / g Zml. 3 shows cytotoxic activity against cell carcinoma line A704. ♦: only cisplatin, fraction: coexistence of cisplatin and 0.1 lz g / ml of IL-6 antibody or IL-6R antibody, ▲: coexistence of cisplatin and 1 gZml of IL-6 antibody or 1L-16R antibody The lower column shows the cytotoxic activity (%) in the presence of cisplatin and 10 g Zml of IL-16 antibody or 16R antibody.

Figures 6A and 6B show patients with vigorous coexistence of cisplatin at 0, 0.1, 1 or 10 g / ml with 1L-6 antibody (Figure 6A) or 16R antibody (Figure 6B). 1 shows the cytotoxic activity against fresh renal cell carcinoma obtained from FIG. ♦ indicates only cisplatin, 顬 indicates coexistence of 0.1 g Zml of IL-6 antibody or IL-6R antibody, and ▲ indicates coexistence of 1 / g Zml of IL-6 antibody or IL-6R antibody. The lower column shows cytotoxic activity (%) in the presence of cisplatin and 10 / gZml of IL-6 antibody or 1L-6R antibody. .

Figures 7A and 7B show the results when 0, 0.1, 1 or 10 µg Zml of cisplatin and IL-6 antibody (Fig. 7A) or IL-6R antibody (Fig. 7B) coexist vigorously. Cytotoxicity against fresh renal cell carcinoma obtained from patient 2 Show activity. ♦ indicates the presence of only cisplatin, and Xin indicates the presence of 0.1 gZm of cisplatin; L-6 antibody or IL-6R antibody. ▲ indicates the presence of 1 agZml of IL-16 antibody or IL-6R antibody of cisplatin. In the figure, Okina shows the cytotoxic activity (%) in the presence of cisplatin and 10 / g ml of IL-6 antibody or IL-6R antibody.

Figures 8A and 8B show patients with coexisting 0, 0.1, 1 or 10 µg Zml of cisplatin and IL-16 antibody (Figure 8A) or IL-6R antibody (Figure 8B). 3 shows the cytotoxic activity against fresh renal cell carcinoma obtained from 3. ♦ indicates only cisplatin, 鼴 indicates coexistence of cisplatin and 0.1 g of IL-6 antibody or IL-6R antibody, and ▲ indicates that of cisplatin and 1 / zg Zml of IL-16 antibody or IL-16R antibody. Hata shows the cytotoxic activity (%) in the presence of cisplatin and 10 g / ml of IL-16 antibody or IL-6R antibody.

FIGS. 9A and 9B show 肾 cell carcinoma in the presence of lipoplatin at 0, 1, 10 or 100 g Zml at the concentration of —6 antibody (FIG. 9A) or IL-6R antibody (FIG. 9B). Shows cytotoxic activity against strain Caki-1. ♦ indicates only carbobratin, and Japan indicates the presence of 0.1 lg Zml of I-6 antibody or IL-6R antibody in the country. ▲ indicates 1 ug / ml of 6 antibody or IL-6R antibody with carboplatin. In the coexistence, Hata shows the cytotoxic activity (%) in the coexistence of calpoplatin and 10 8 11) 1 1-6 antibody or IL-16R antibody.

A in FIG. 10 indicates GST-in renal cell carcinoma cell line Caki_1 treated with culture medium (control), 10iz g Zml of cis-bratin, 10 / g Zml of IL-16 antibody or IL-16R antibody. FIG. 7 shows the results of Northern blotting of total Caki-1 RNA using a GST-ττ cDNA probe for examining 7Γ mRNA expression. Lane 1 was treated with culture medium only, lane 2 was treated with cisplatin, lane 3 was treated with IL-6 antibody, and lane 4 was treated with 1-6R antibody . B in FIG. 10 is a diagram of a gel used for Northern plots, which was stained with ethidium umide. Each lane indicates the presence of RNA. Specific description

The antitumor agent-enhancing agent comprising 1L-6 antagonist of the present invention enhances the antitumor effect when used in combination with an antitumor agent in treating a tumor. In addition, it has the effect of reducing the required dose of an antitumor agent and increasing the sensitivity of the antitumor agent even to a treatment-resistant tumor for which a therapeutic effect is not recognized by ordinary chemotherapy.

The anti-tumor agent whose antitumor effect is enhanced by the action enhancer of the present invention is a chemotherapeutic agent that acts on tumor cells to suppress the growth and proliferation of tumor cells and has a therapeutic effect on tumors. Chemotherapeutic agents include alkylating agents, metabolic antagonists, antitumor antibiotics, plant-derived alkyloids, formone therapeutic agents, and platinum compounds. Among such antitumor agents, a platinum compound having an antitumor effect of mitomycin C ゃ, which is an antitumor agent, is particularly preferred. Platinum compounds have a platinum atom and form a complex with other atoms. By binding to DNA, it inhibits the synthesis of DNA in tumor cells, inhibits the division of tumor cells, and has an antitumor effect. To date, platinum compounds having antitumor activity include cisplatin (cis-diamminedich1 orop1 atinum (11), having the following structural formula).

NH, NO, C1 NH, NO, C1 canolehobratin (cisd i ammine (1, 1-cycl obu taned i carboxy 1 ato;-Platinum (II), having the following structural formula),

254-S ((Glycolato-0, 0 ') d i ammi nep 1 a t i num, having the following structural formula),

DWA-2114R ((-)-(R) -2 -am inomethyl pyrrol idine (1, 1-cyclobutane di carboxylatoplat inum (11), having the following structural formula),

NK-121 ((R) -cis-2-methy 1 -1,4-butane.diamine (1, 1-cyclobutaned icarboxylato) plat inum (II), having the following structural formula),

Okisariburachin (oxaliplatin; USAN, Oxalato (trans -1, 2-cyc lohexanediamine) platinum (11), have a structural formula below ^_ 9),

TRK-710 ((alpha-acetyl-gamma-methyl tetronate) z- (I-1, 2-diaminocyclohexane) platinum (II), having the following structural formula)

Etc. are known. Among these platinum compounds, cisplatin, carboplatin and DWA-2114R are particularly preferred.

These antitumor agents are formulated in a usual manner. For example, in the case of a platinum compound, if necessary, it is mixed with an adjuvant and a simple substance used as a drug, and used orally or parenterally, preferably as an injection. Can be In the case of an injection, it is advisable to mix it with distilled water or a salt solution such as sodium chloride or potassium chloride, a glucose solution or a physiological saline solution. The amount of the antitumor agent in these preparations is desired to be a convenient unit dose depending on the age and condition of the patient. For example, when used for the treatment of tumors in adults, 10 to 2000 mgZm once a day ² (volume area) may be administered, and depending on the dose, continuous administration for 5 days or a drug holiday of 114 weeks between administrations may be provided.

Tumor cells having an antitumor effect by the action enhancer of the present invention are tumors having IL-6R and exhibiting proliferation and Z or treatment resistance using IL-6 as one physiologically active substance. . Such tumors include renal cell carcinoma (Miki, S. et al., FEBS Letter, 250, 607-610, 1989) and myeloma (Kawano, M. et al., Nature, 332, 83-85, 1988). ), Ovarian cancer (Kobayash i, H. et al., Proceedings of the 53rd Annual Meeting of the Japanese Cancer Society, 271 pages, 874, 1994), EB virus-infected B lymphoma (Tosata, G. et al., J. Virol. 64, 3033-3041, 1990), adult T-cell leukemia (Sawada, T. k Takatsuki, K. Br. J. Cancer, 62, 923-924, 1990), prostate cancer (Siegall, CB et al., Cancer Res. , 50, 7786-7788, 1990) and Kaposi sarcoma (Miles, S.A. et al., Proc. Natl. Acad. Sci. 87, 4068-4072, 1990).

The IL_6 antagonist used in the present invention may be of any origin as long as it blocks signal transmission by IL-6 and inhibits the biological activity of 1L-16. As an IL-6 antagonist, IL-6 Body, IL-6R antibody, gpl30 antibody, IL-6 variant, antisense oligonucleotide of IL-6R, partial peptide of IL-16 or IL-6R, and the like.

Antibodies used as 1L-16 antagonists in the present invention, for example, IL-6 antibody, IL-6R antibody, or gpl30 antibody may be of any origin and type (monoclonal, polyclonal). In particular, a monoclonal antibody derived from a mammal is preferred. By binding to IL-6, IL-6R or gpl30, these antibodies inhibit the binding of IL-6 to IL-6R or IL-6R and gpl30, block 1L_6 signaling, and Is an antibody that inhibits the biological activity of

The animal species of the cell producing the monoclonal antibody is not particularly limited as long as it is a mammal, and may be derived from a human antibody or a mammal other than human. As a monoclonal antibody derived from a mammal other than human, a monoclonal antibody derived from a rabbit or a rodent is preferred because of its ease of preparation. The rodent is not particularly limited, but mouse, rat, hamster and the like are preferably exemplified.

Such antibodies include MH166 antibody (Matsuda et al., Eur. J. Immunol. 18: 951-956, 1988) and SK2 antibody (Sato et al., 21st Annual Meeting of the Immunological Society of Japan). Scientific Records, 21: 116, 1991). Examples of the IL-6R antibody include PM-1 antibody (Hirata et al., J. Immunol. 143: 2900-2906, 1989), AUK12-20 antibody, AUK64-7 antibody, and AUK146-5-15 antibody (International Patent Application Publication). Among them, the IL-6R antibody is particularly preferable, and specific examples thereof include the above-mentioned PM-1 antibody.

A monoclonal antibody can be basically produced using a known technique as follows. That is, IL-6, IL-6R or gpl30 It is used as a sensitizing antigen, and is immunized according to a conventional immunization method, and the obtained immune cells are fused with a known parent cell by a normal cell fusion method, and are then subjected to a normal screening method. It can be prepared by screening monoclonal antibody-producing cells.

More specifically, a monoclonal antibody can be prepared as follows. For example, the sensitizing antigen is preferably a human-derived antigen. In the case of human IL-6, the human sensitizing antigen is a human IL-16 which is disclosed in Hirano et al., Nature, 324: 73, 1986. It is obtained by using gene sequences. After the human 6 gene sequence is inserted into a known expression vector system to transform an appropriate host cell, the desired IL-6 protein is isolated from the host cell or the culture supernatant. After purification, the purified IL-6 protein may be used as a sensitizing antigen.

In the case of human IL-16R, an IL-6R protein can be obtained by using the gene sequence disclosed in European Patent Application Publication No. EP325474 and following the same method as for human IL-16. There are two types of IL-6R: those expressed on the cell membrane and those soluble off the cell membrane (sIL-6R) (Yasukawa et al., J. Biochem., 108, 673-676, 1990). . sIL-6R is composed mainly of the extracellular region of IL-16R bound to the cell membrane, and differs from membrane-bound IL-16R in that the transmembrane region or the transmembrane region and the intracellular region are defective. Is different.

In the case of human gpl30, gpl30 protein can be obtained by using the gene sequence disclosed in European Patent Application Publication No. EP411946 and following the same method as for IL-6.

The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. For this, mice, rats, hamsters, egrets, etc. are used. Immunization of an animal with a sensitizing antigen is performed according to a known method. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, sensitizing antigen

After diluting and suspending an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, etc., add an appropriate amount of a normal adjuvant, for example, complete Freund's adjuvant, and emulsify the suspension. It is preferred to administer several doses every 4 to 21 days. In addition, an appropriate carrier can be used at the time of immunization with the sensitizing antigen.

After immunization in this way and confirming that the level of the desired antibody in the serum is increased, immune cells are removed from the mammal and subjected to cell fusion. Preferred immune cells are particularly preferred. Splenocytes. Mammalian myeloma cells as the other parent cells fused with the immune cells are already known in various cell lines, for example, P3 (P3x63Ag8.653) (J. Immunol. 123: 1548, 1978), p3-Ul (Current Topics in Micro-biology and Immunology 81: 1-7, 1978), NS-1 (Eur. J. Immunol. 6: 511-519, 1976), MPC-11 (Cell, 8: 405-415, 1 976), SP2 / 0 (Nature, 276: 269-270, 1978), F0 (J. Immunol. Met h. 35: 1-21, 1980), S194 (J. Exp. Med) 148: 313-323, 1978), R210 (Nature, 277: 131-133, 1979) and the like are preferably used.

Cell fusion between the immune cells and myeoma cells is basically performed according to a known method, for example, the method of Milstein et al. (Milstein et al., Methods Enzymol. 73: 3-46, 1981). It can be carried out.

More specifically, the cell fusion is performed, for example, in a normal nutrient culture in the presence of a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and if necessary, an auxiliary agent such as dimethyl sulfoxide is added to enhance the fusion efficiency. Can also be used. The ratio of the use of the immune cells to the myeloma cells is preferably, for example, 11 to 10 times the number of the immune cells to the myeoma cells. Examples of the culture medium used for cell fusion include RPM11640 culture medium and MEM culture medium suitable for the proliferation of the myeloma cell line, and other ordinary cultures used for cell culture of this type. A fluid can be used, and a serum replacement fluid such as fetal calf serum (FCS) can also be used in combination.

For cell fusion, a predetermined amount of the immune cells and myeloma cells is mixed well in the culture medium, and a PEG solution previously heated to about 37 ° C., for example, an average molecular weight of about 1,000 to 6,000 The desired fused cells (hybridoma) are formed by adding and mixing the PEG solution of the above at a concentration of 30-60% (wZv). Subsequently, by repeating the operation of successively adding an appropriate culture solution and centrifuging to remove the supernatant, a cell fusion agent or the like unfavorable for the growth of the hybridoma can be removed.

The hybridoma is selected by culturing it in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminobuterin and thymidine). Culture in the HAT culture medium is continued for a period of time sufficient to kill cells other than the target hybridoma (non-fused cells), usually several days to several weeks. Next, a conventional limiting dilution method is performed, and screening and monocloning of the hybridoma producing the desired antibody are performed.

The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture medium, and can be stored for a long time in liquid nitrogen. .

To obtain a monoclonal antibody from the hybridoma, a method of culturing the hybridoma according to an ordinary method and obtaining the culture supernatant, or transferring the hybridoma to a mammal compatible therewith Injection, proliferation and ascites are used. You. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.

Monoclonal antibodies include not only those obtained from hybridomas produced by cell fusion of antibody-producing cells obtained by immunization with an antigen, but also those obtained by cloning an antibody gene and incorporating it into an appropriate vector. Can be used as a monoclonal antibody produced by using a gene recombination technique by introducing the gene into a known cell line, for example, COS, CH0, etc. (for example, Vandamme, A-M. Et al., Eur J. Biochem., 192, 767-775, 1990).

Furthermore, the monoclonal antibody obtained by the above-mentioned method can be purified to a high degree of purity using ordinary purification means such as a salting-out method, a gel filtration method, and an affinity chromatography method. It can be purified.

The monoclonal antibodies thus prepared can be obtained by ordinary immunological means such as radioimmunoassay (RIA), enzyme immunoassay (E1A, ELISA), and immunofluorescence assay (Immunofluorescence analysis). It can be confirmed that the antigen is recognized with high sensitivity and high accuracy.

The monoclonal antibody used in the present invention is not limited to the monoclonal antibody produced by the hybridoma, but may be artificially modified for the purpose of, for example, reducing the antigenicity to humans. What you do is better. For example, a chimeric antibody comprising a variable region of a mammalian non-human, eg, mouse, monoclonal antibody and a constant region of a human antibody can be used. It can be produced using known methods for producing chimeric antibodies, particularly using gene recombination techniques.

Furthermore, a reshaped human antibody can be used in the present invention. This is obtained by transplanting the complementarity-determining regions of a mammal other than a human, for example, a mouse antibody, into the complementarity-determining regions of a human antibody. Is also known. Using the known method, a reshaped human antibody useful in the present invention can be obtained, and a preferred example thereof is a reshaped PM-1 antibody (for example, an international patent) (See Application Publication No. W092-19759).

If necessary, amino acid in the framework (FR) region of the variable region of the antibody may be replaced so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen binding site. Good (Sato et al., Cancer Res. 53: 1-6, 1993). In addition, antibody fragments, such as F (ab ') ₂ , Fab or Fv, and Fv of H and L chains, with appropriate linkers, as long as they bind to antigen and inhibit the activity of IL-6 A gene encoding the ligated single-glutinin Fv (scFv) can be constructed, expressed in a suitable host cell, and used for the aforementioned purpose. (See, e.g., Bird et al., TIBTEC H, 9: 132-137, 1991).

scFv is formed by linking the H chain V region and L chain V region of an antibody. In this sc Fv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883, 1988). The H chain V region and L chain V region in scFv may be derived from any of the antibodies described above. These V regions are preferably connected by peptide linkers. As the peptide linker, for example, any single-chain peptide consisting of amino acid residues 12 to 19 is used.

4

The scFv-encoding DNA is a DNA encoding the H chain or the H chain V region of the antibody, and a DNA encoding the L chain or the L chain V region. The DNA portion encoding the desired amino acid sequence is amplified by the PCR method using a pair of primers defining both ends, and then a portion of the peptide linker is also encoded. It is obtained by combining and widening a pair of primers that define DNA and its both ends so as to be linked to an H chain and an L chain, respectively.

Further, once DNAs encoding scFv are prepared, an expression vector containing them and a host transformed by the expression vector can be obtained according to a conventional method. The scFv can be obtained according to a conventional method. The scFv is superior to the tissue transferability of the antibody molecule, and is expected to have the same function as the reshaped human antibody.

The IL-16 variants used in the present invention include those disclosed in Brakenhoff et al., J. Biol. Chem. 269: 86-93, 1994 or Savino et al., EMBO J. 13: 1357-1367, 1994. Is mentioned.

As a modified IL-6, by introducing a mutation such as substitution, deletion, or human mutation into the amino acid sequence of IL-6, the IL-6R can maintain its IL-6R binding activity while maintaining its IL-6R binding activity. One that does not have a signal transduction action is used. Further, 1L-6 as its origin is not limited to animal species as long as it has the above properties, but it is preferable to use human origin in consideration of antigenicity. Specifically, the amino acid sequence of IL-16 can be obtained by using a known molecular modeling program such as WHATIF (Vriend et al., J. Mol. Graphics, 8: 52-56, 1990). This is done by predicting the secondary structure and assessing the overall effect of the mutated amino acid residue. After determining the appropriate mutant amino acid residue, the vector containing the nucleotide sequence encoding the human IL-16 gene is used as a template for PCR (polymerase chain reaction). By introducing a mutation by the method, a gene encoding a 1L-6 variant can be obtained. This is inserted into an appropriate expression vector if necessary, expressed in E. coli cells or mammalian cells, and isolated or purified as it is contained in the culture supernatant or by a conventional method. Binding activity to IL-6 and IL-6 The neutralizing activity of the signal transmission can be evaluated.

The IL-16 partial peptide or IL-6R partial peptide used in the present invention binds to IL-6R or IL-6, respectively, as long as it has no IL-16 activity transfer action. Regardless of the sequence of the fragment. See US Patent Publication US5210 075 and European Patent Publication No. 6-1126 for IL-6 and IL-6R partial peptides. For the IL-6R antisense oligonucleotide, see Japanese Patent Application No. 5-300338.

The antitumor agent potentiator comprising the IL-6 antagonist of the present invention has an IL-16R as long as it blocks 1L-6 signal transduction and captures and enhances the action of the antitumor agent. IL-6 can be effectively used as a physiologically active substance for the treatment of any tumor that shows growth and no resistance or treatment resistance.

The potentiator of the antitumor agent comprising the IL-16 antagonist of the present invention is preferably administered parenterally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. It can be administered steadily. Furthermore, it can be in the form of a pharmaceutical composition kit with at least one pharmaceutical carrier or diluent.

The dose of the antitumor agent potentiator comprising IL-16 antagonist of the present invention to a human varies depending on the patient's condition, age and administration method, but it is necessary to select an appropriate amount as appropriate. For example, in the case of IL-6R antibody, no more than four divided doses can be selected in a range of approximately 11 mg / l mg of patients. It can also be administered at a dose of 11 mg / kgZweek. However, the dose of the antitumor agent potentiator comprising the IL-6 antagonist of the present invention is not limited to these doses.

Antitumor agent action enhancer comprising 16 antagonist of the present invention Can be formulated according to a conventional method (Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Boston, USA). For example, injectable preparations are prepared by dissolving purified IL-16 gonist in a solvent such as physiological saline, buffer, glucose solution, etc., and adding an anti-adsorption agent such as Tween80, gelatin Or serum albumin (HSA) or lyophilized for reconstitution before use. As an excipient for freeze-drying, for example, sugar alcohols and sugars such as mannitol and glucose can be used.

Example

Hereinafter, the present invention will be described specifically with reference to Examples, Reference Examples, and Experimental Examples, but the present invention is not limited thereto.

Example 1 Effect of IL-16 Antibody or IL-6R Antibody on Tumor Cell Sensitivity to Antitumor Agent

The effect of -6 antibody or IL-6R antibody on the sensitivity of tumor cells to various antitumor agents was examined.

(1) Preparation of human renal cell carcinoma

Human renal cell carcinoma cell lines Caki-1 and Caki-1 are sublines of cis-bratin-resistant cell line Caki-1 ZDDP, human cell carcinoma ACHN, and human renal cell carcinoma A704 (Giard, DJ et al., J. Nat. 1. Cancer Inst. 51, 1417-1423, 1973) was added to 25 mM HEPES, 2 mM L-octylamine, 1% non-essent ial amino acid, 100 U Zml penicillin, 100 g / ml Streptmycin and 10% heat-inactivated. In a RPMI 1640 culture medium (above, manufactured by Gibco) containing fetal serum (FBS) (hereafter referred to as complete culture medium), on a plastic dish. The cells were cultured to form a monolayer.

On the other hand, the method of Mizutani, Y. et al. (Cancer 69, 537-545, 1992) was used. Accordingly, neoplastic tumor cells were obtained from renal cell carcinoma patients. Upon surgical treatment of three patients with renal cell carcinoma, renal cell carcinoma tumor tissue was obtained. After confirmation of 肾 cell carcinoma by histological classification, the tumor tissue was finely decomposed with SmgZml collagenase (manufactured by Sigma Chemical Co.) to prepare a tumor cell suspension. After three washes with RPMI 1640 medium, the cell suspension was placed on a discontinuous gradient consisting of 2 ml each of 100%, 80% and 50% fico-hypaque in a 15 ml plastic tube. The layers were overlaid and centrifuged at 400 × g for 30 minutes. Remove the lymphocyte-enriched monocyte layer from the 100% layer and obtain tumor cells and mesothelial cells from 80% eyebrows.To prevent contamination with other cells, complete culture in a 15 ml plastic tube. The cell suspension, enriched in tumor cells, was overlaid on a discontinuous gradient consisting of 3 ml each of 25%, 15%, 10% Percoll and centrifuged at room temperature at 25 × g for 7 minutes. Tumor cells separated from lymphocytes were obtained from the bottom of the tube. After the obtained tumor cells were washed and suspended in a complete culture solution, the survival of the tumor cells was confirmed by trypan blue staining. These tumor cells thus prepared were used in the following experiments.

(2) Confirmation of IL-6 production in renal cell carcinoma by ELISA

IL IL-6 is present in the culture supernatant of cell tumor cell lines Caki-1 and Caki-1 / DDP, ACHN, A704 and 704 cell carcinoma patients (No. 1 to 3). Was assayed by the BLISA (enzyme-linked immunosorbent assay; method).

A 96-well EUSA plate was supplemented with 100-1 IL-16 antibody, and the ELISA plate was coated with the IL-6 antibody at least. These plates were stored at 4 ° C for up to 4 weeks before use. Plates coated with IL-16 antibody were washed three times and blocked for 1 hour with ELISA PBS containing 1% BSA (perforated serum albumin). After two washes, Escherichia coli-derived recombinant IL-16 (Yasukawa et al., Biotechnol. Lett., 12, 419, 1990) was added to each pellet as a 100 ^ 1 tumor cell culture supernatant or control.

Plates were incubated for 1 hour, washed three times, and reconstituted with 100 μl of each anti--16 polyclonal antibody (Matsuda, T. et al., Eur. J. Immunol., 18, 951-956, 1988). Was added. Plates were incubated for 1 hour, Alkaline phosphatite-conjugated goat anti-heron G was added to each well and incubated for an additional hour. Plates were washed and incubated with anorecalophosphate substrate (Sigma 104, Sigma Chemical Co.). Two hours later, the absorbance at 405 nm was measured using EL1SA READER (Immunoreader, manufactured by Japan Intermed Co. ltd.). The results revealed that these renal cell carcinomas produced IL-6. (See Table 1).

table 1

IL-6 concentration in supernatant

RCC cells IL-1-6 degree (pgZml; average soil standard deviation)

Caki-1 1337 ± 35

Caki-1 DDP 3900 ± 325

A704 1290 Sat 141

ACHN 1282 ± 106

Fresh RCC cells (Patient No. 1) 1236 ± 71

Fresh RCC cells (Patient No. 2) 42 ± 4

Fresh RCC cells (Patient No. 3) 2579 ± 219

(3) Effect of 16 antibody or IL-16R antibody on cytotoxicity of antitumor agent

Various concentrations of antitumor agents, ie, cisplatin (cis-diamminedi chloroplatinum (II)). mitomycin C (MMC), adriamycin (ADR), vinblastine (VBL) and 5 — phnoreolouracinole (5 — IL-6 antibody or 1L on the sensitivity of each renal cell Caki-1, Caki-1 / DDP, ACHN, A704 and fresh tumor cells from the patient (No. 1-3) to F. luorouraci 1; To examine the effect of the 6R antibody, the MTT method (Mizutani, Y. et al., Cancer 73, 730-737, 1994) was performed.

100 1 of the above renal cell carcinoma tumor cell suspension (2 × 10 ⁴ cells) was dispensed into a 96-well bottom microtiter plate (Corning Glass Works, Corning). The plates at 37 ° C, placed under 5% C0 ₂ present humidified, and the tumor cells were cultured for 24 hours. The cell culture supernatant was aspirated off and the tumor cells were washed three times with RPMI 1640 medium. IL-6 antibody (Matsuda, T. et al., Eur. J. Immunol., 18, 951-956, 1988) or IL-6R antibody (Hirata, Y. et al., J. Immunol., 143, 2900-2906, 1989) ), A complete culture solution was added to each well as a solution or control containing 200% of each antitumor agent, and cultured at 37 ° C for 24 hours. 20 ^ 1 of MTT solution (manufactured by Co. 5 mg / mK Si ma Chemical ) was added to each Ueru, with continued 37 ° C, and incubated for 4 hours at 5% C0 ₂ present humidified under. The culture solution was removed from each bottle and replaced with isopropanol containing 0.05N HC1 (manufactured by Sigma Chemical Co.).

The absorbance at 540 nm of each gel solution was measured with a microculture plate reader (Immunoreader, manufactured by Japan Intermed Co. ltd.). The percentage of cytotoxicity was calculated by the following formula. Cytotoxicity (%) =

(1-(Absorbance of experimental group: Absorbance of control group)) χιοο As a result, in Caki-1 cells, cisbratin (see Fig. 1A, B) or MMC (Fig. A, B) clearly increased the cytotoxicity of the control antibody M0PC 3 ZC (J. Natl. Cancer In the experimental group to which Inst. (Bethesda), 41, 1083, 1968) was added, there was no effect on the sensitivity to cissplatin or related C. Compared to the case where the antitumor agent is added alone, the required amount of the antitumor agent is 1 Z10-1 to find the same level of cytotoxic effect in the presence of the antitumor agent and IL-16 antibody or IL-6R antibody. Z100. On the other hand, in the co-presence of IL-6 antibody or IL-16R antibody with ADR, VBL or 5-FU, the sensitivity of tumor cells to antitumor agents did not change.

The resistance of Caki-1 / DDP cells to cisplatin was overcome by the coexistence of IL-16 antibody or IL-6R antibody and cisbratin (see FIGS. 3A and B). Such synergy between IL-6 antibody or IL-6R antibody and cisplatin was demonstrated by other 肾 cell carcinoma lines ACHN (see FIGS. 4A and B), A704 (see FIGS. 5A and B) and The same was observed in neoplastic tumor cells obtained from patients (see A and B in Figs. 6, 7, and 8).

Example 2 Effect of IL-16 Antibody or IL-16R Antibody on Renal Cell Carcinoma Sensitivity to Carbobratin

Using the renal cell carcinoma cell line Caki-1, the cytotoxicity of carboplatin prepared at various concentrations and the IL-16 antibody or the 16R antibody was examined in the same manner as in Example 1 above. As a result, the sensitivity of Caki-11 cells to carbobratin was enhanced (see FIGS. 9A and B).

Example 3 IL-16 antibody or IL-1 related to intracellular accumulation of antitumor agent

Effect of 6R antibody

10 zg / ml cisplatin or 100 z gZml of 5—FU and control culture, 10 g "ml of control antibody M0PC3 / C, 10 / z gZml of 1L-16 antibody Alternatively, Caki-1 cells were cultured for 24 hours in the presence of the IL-16R antibody combination.

The medium is then removed and the cells are washed three times with RPMI 1640 medium did. The intracellular accumulation of cisplatin was determined by the frameless atomic absorption spectrometry method (Daley-Tates, PT et al., Biochem. Pharmacol., 34, 2263-2369; Riley, CM et al., Analytical Biochem., 124, 167). -179, 1982). As a measuring instrument, Zeeman Z-8000 (Zeeman z-8000 Spectrophotometer, manufactured by Hitachi Co. Ltd.) was used. The intracellular accumulation of 5-FU was measured by gas chromatography-mass spectrometry (Marunaka, T., et al., J. Pharm. Sci., 69, 1296-1300, 1980). As a measuring instrument, a JMS-D 300 mass spectrometer (manufactured by J0EL) equipped with a JGC-20KP gas chromatograph was used. The results are shown in Table 2. The IL-6 or IL-6R antibody was found to have no effect on the accumulation of cisplatin, 5-FU in tumor cells.

Table 2

Intracellular accumulation of CDDP or 5-FU (ngZlO ⁷ cells)

Drug treatment

Control (Culture medium) Control Ab Anti-IL-6mAb Anti-IL-6R mAb CDDP 0.28 ± 0.05 0.27 ± 0.02 0.26 ± 0.05 0.27 ± 0.02 5—FU 1.48 ± 0.32 1.57 ± 0.45 1.50 ± 0.19 1.63 ± 0.31 Numerical values of three experiments Calculated from average data (average soil standard deviation)

Example 4 Expression of Glutathione S-transf erase- (GST-π)

Effect of cisplatin, IL-6 antibody or 16R antibody on Caki-1 cells as control medium, 10 g Zml cisplatin, 10 μg / ml IL-6 antibody or IL-1R The cells were cultured with the 6R antibody for 4 hours. Then, the total RNA of the cells was prepared by the method of Mizutani, Y, et al. (Cancer 73, 730-737, 1994), and 200 mM mops (M0PS; 3-[N-morpholy) was used to obtain 10 g RNAZ lanes. [No] propanesulfonic acid), 50 mM sodium acetate and 10 mM EDTA sodium The gel was electrophoresed on a 1.2% agarose-2.2M HCH0 gel in 1x MOPS buffer. Then, the RNA was transferred to Biodyne A membrane (Poll) in a 20X SSC solution containing 3 M NaCl and 0.3 M sodium citrate (pH 7.0). 50—100 ng of GST—7Γ cDNA probe (Nakagawa, K. et al., J. Biol. Cem. 265, 4296-4301, 1990) labeled with ³² P—dCTP (NEN) using the random oligo primer extension method did. The nylon membrane onto which the RNA was transferred was cross-linked with ultraviolet light and hybridized with the probe. The results are shown in FIG.

Cisplatin did not affect GST-7Γ mRNA expression in Caki-1 cells. However, the addition of IL-6 antibody or IL-6R antibody reduced the expression of GST-7 mRNA. These facts suggest that the decrease in the expression level of GST-7Γ mRNA is involved in increasing the sensitivity of renal cell carcinoma to cisplatin by the IL-6 antibody or the 16R antibody. Industrial applicability

By coexisting an IL-16 antagonist such as an IL-6 antibody or an IL-6R antibody with the antitumor agent, the sensitivity of the tumor cell to the antitumor agent was observed at a lower dose, and the IL-16 antagonist was inhibited by the antitumor agent. It is confirmed that the combination results are exhibited by the tumor agent. Furthermore, it is demonstrated that tumor cells that are resistant to treatment with an antitumor agent can be treated as a result of enhancing the sensitivity of the antitumor agent to IL-16 antagonist.

In addition, the potentiator of the antitumor agent comprising IL-6 antagonist of the present invention can reduce the toxicity of the antitumor agent to tissues by reducing the required dose of the antitumor agent. Therefore, it is expected as an action enhancer of an antitumor agent.

Claims

The scope of the claims

1. An antitumor agent action enhancer comprising interleukin 6 (IL-6) antagonist.

2. The action enhancer according to claim 1, wherein the IL-6 antagonist is human IL-6 antagonist.

3. The action enhancer according to claim 1 or 2, wherein the IL-6 antagonist is a monoclonal antibody.

4. The action enhancer according to claim 3, wherein the IL-6 antagonist is a 1L-6 antibody or an IL-16 receptor antibody.

5. The potentiator of claim 4, wherein the one antagonist is a PM-1 antibody.

6. The action enhancer according to claim 5, wherein the 1L-6 antagonist is a humanized PM-1 antibody.

7. The action enhancer according to any one of claims 1 to 6, wherein the antitumor agent is a chemotherapeutic agent.

8. The activity enhancer according to claim 7, wherein the antitumor agent is a platinum compound having an antitumor effect.

9. The action enhancer according to claim 7, wherein the antitumor agent is mitomycin C.

10. The action enhancer according to claim 8, wherein the antitumor agent is selected from the group consisting of cisbratin, carboblatin, 254-S, DWA-2114R or NK-121.

11. The tumor whose antitumor effect is enhanced is a tumor having IL-16 receptor and showing proliferation and Z or treatment resistance using IL-16 as a bioactive substance. Action enhancer.